Urine: a place where HEV cannot hide!
Authors: Patrick Behrendt, Jérôme Gouttenoire
Journal: Hepatology (2023)

Hepatitis E results from an infection by a positive-strand RNA virus, namely HEV, of which four main humanopathogenic genotypes (GTs) exist. While the virus is recognized to cause waterborne endemic outbreaks in particular in parts of Africa and Asia (GT1 and GT2) since the eighties, new light was shed on HEV when discovered as widely circulating in industrialized countries, where zoonotically transmitted GT3 and 4 are present.1 GT1 and GT2 are believed to cause solely acute infections and display particularly high mortality rates in pregnant women. In general, GT3-related and GT4-related disease is believed to be less virulent and mainly individuals with underlying liver disease are at increased risk to develop a severe disease. One hallmark of GT3 and 4 infections are chronic disease courses that can occur in up to 60% of immunosuppressed individuals with the risk for a rapid progression to advanced liver fibrosis.2 Currently, no direct antiviral treatment is approved. However, in case of chronic infections, international guidelines recommend treatment with ribavirin for 3-6 months which leads to clearance in ~85% of the cases.3 While diagnostic of acute HEV infection is relying mostly on serological markers (anti-HEV specific immunoglobulin M) detection of HEV RNA in plasma and stool remains the gold-standard due to its higher sensitivity and specificity and is irreplaceable for diagnosis in immunosuppressed individuals.4 During infection, the ORF2 capsid protein represents the main viral antigen (Ag) produced, not only, as nonglycosylated form assembling the viral capsid of infectious particles, but also in large amounts as glycosylated Ag secreted in plasma. This latter ORF2 form is believed to serve as immunological decoy.5,6 In the study published in this issue of Hepatology,7 the authors identified the presence of the HEV ORF2 capsid protein as being an early and reliable marker of HEV infection in patients but also in an animal model as the HEV Ag is eliminated through the kidney in the urine. Interestingly, the level of HEV ORF2 Ag in the urine is about 10-fold higher than in serum which also translates to an earlier detection than in blood. Results in animal model suggest that HEV Ag in urine is at least as sensitive as HEV RNA detection in stool. In addition, qualitative analyses revealed that the Ag is found as a cleaved form of about 20 kDa corresponding to a C-terminal portion of HEV ORF2 protein, known as P-domain. Using a panel of monoclonal antibodies targeting different epitopes of HEV ORF2 protein as well as investigating the behavior of ORF2 mutants in an animal model, the authors identified a cluster of amino acid residues exposed at the surface of ORF2 P-domain which may be engaged in interaction with a specific cell receptor to transfer of the viral Ag from the blood into the urine. The findings of this study possibly will have strong implications for the diagnostic and the clinical management of hepatitis E patients in the future. Given the sensitivity of ORF2 protein detection in urine of about 95% as presented in this study,7 a rapid test based on the detection of the viral Ag in an easy-to-collect sample, such as urine, may greatly help diagnosing this viral infection in a timely manner especially in minimal laboratory settings. Although sensitivity of urine HEV Ag as marker of infection has been delineated previously in smaller cohorts,8,9 its surprisingly early detection as compared to other markers of infection points to a valuable test for diagnosis of HEV infection at an early stage. While the results presented in the study7 hold promises, further investigations extended to larger cohorts of patients need to assess the diagnostic value of the urine HEV Ag, in regard of various parameters such as serology status, viral GT, clinical presentation including symptoms or chronic infection. Indeed, future studies should prove the accurate detection of urine HEV Ag for other viral species including the recently emerging rat HEV and the GT1 and 2 found in developing countries. Not only, that it may be easily and timely implemented in resource-limited regions, detection of HEV Ag in urine may offer the opportunity to draw a timelier picture of the viral spread and to react with immediate safety-measures in outbreak situation. Furthermore, earlier access to clinical care for patients at risk for a severe course of infection—for example pregnant women—could be provided. In some circumstances, patients with underlying liver cirrhosis or advanced fibrosis may be referred to a transplant-center late in the course of infection to be evaluated for liver transplantation. Most often, in such cases HEV diagnosis has been made based on serological markers. However, as sometimes unhealthy alcohol consumption cannot immediately be ruled out as possible mechanism which causes the acute/acute-on-chronic liver injury, a direct measure of infection is desirable. Due to the rather short viremic phase, HEV RNA detection in particular in plasma may already show a negative result. Hence, the prolonged release of viral capsid in the urine may support the diagnosis of HEV in these circumstances and thereby facilitate implementation of appropriate treatment measures, for example, liver transplantation, without any unnecessary delay. Another application may be the guidance of antiviral therapy of chronic hepatitis E with ribavirin. Being HEV RNA negative in the stool at end of treatment seemed to be a reliable marker to anticipate sustained virological response.3,4 However, enhanced sensitivity of current analysis methods leads to increased detection of HEV RNA at end of treatment, including in patients for which the treatment would be prolonged without any benefit but with the risk for developing severe adverse-effect due to ribavirin.10 Therefore, urine HEV Ag needs to be evaluated as possible superior marker for guidance of therapy in the future. The case of the chronically infected patients is particularly interesting as they may likely shed continuously the ORF2 Ag in the urine. In addition, to the rapid aggravation of the liver functions in those patients, the consequences of the long-term shedding of the viral Ag through the kidney shall be addressed because it may play a relevant role for the observed kidney function alteration in infected individuals. So far, presence of HEV ORF2 protein in urine was supposedly linked to infection of the kidney as suggested by the study of Geng et al9 in which HEV RNA and Ag were detected in the urine of acute and chronic hepatitis E patients. The present study7 prove that the detection of HEV RNA and Ag in urine are not necessarily correlated as only 13/47 patients were positive for both and that some presented with very high level of Ag in absence of detectable RNA in the urine. The origin and the mechanisms leading to the secretion of this viral Ag in urine remain still unclear. Although the authors hypothesized that the ORF2 protein is actively transferred through the kidney via a nonidentified cellular receptor, the sequence conservation of such receptor in the animal models used, mouse and rabbit, is questionable and leave open the possibility to other explanation including free transport of small ORF2 fragments across the glomerular barrier. Overall, evidence for the elimination of plasmatic ORF2 capsid protein in the urine provided in the study by Ying et al7 should yield changes in hepatitis E diagnosis but also raise important fundamental questions including related to viral pathogenesis (Figure 1).FIGURE 1: HEV ORF2 antigen eliminated into the urine offers new clinical perspectives. Patients suffering of hepatitis E (HE) are shedding quasi-enveloped HEV (eHEV) in the bloodstream together with large quantities of ORF2 protein. Moreover, HEV ORF2 antigen (Ag) is present in urine of these acutely-infected individuals as small fragments corresponding to the ORF2 P-domain (highlighted in gold). The large amount of urinary HEV Ag and its early and prolonged detection offer several important advantages to be exploited including for diagnostic purposes. HEV ORF2 protein modeling has been prepared on the basis of ORF2 structure (pdb entry 2ZTN) and using VMD1.9.4 software.